The present invention relates to small reaction chambers, such as a reaction chamber including a microarray within a microarray strip, and, in particular, to a method and system for circulating solutions within small sealed reaction chambers.
Microarrays are widely used and increasingly important tools for rapid hybridization analysis of sample solutions against hundreds or thousands of precisely ordered and positioned features on the active surfaces of microarrays that contain different types of molecules. Microarrays are normally prepared by synthesizing or attaching a large number of molecular species to a chemically prepared substrate such as silicone, glass, or plastic. Each feature, or element, on the active surface of the microarray is defined to be a small, regularly-shaped region on the surface of the substrate. The features are arranged in a regular pattern. Each feature may contain a different molecular species, and the molecular species within a given feature may differ from the molecular species within the remaining features of the microarray. In one type of hybridization experiment, a sample solution containing radioactively, fluorescently, or chemoluminescently labeled molecules is applied to the active surface of the microarray. Certain of the labeled molecules in the sample solution may specifically bind to, or hybridize with, one or more of the different molecular species in one or more features of the microarray. Following hybridization, the sample solution is removed by washing the surface of the microarray with a buffer solution, and the microarray is then analyzed by radiometric or optical methods to determine to which specific features of the microarray the labeled molecules are bound. Thus, in a single experiment, a solution of labeled molecules can be screened for binding to hundreds or thousands of different molecular species that together compose the microarray. Microarrays commonly contain oligonucleotides or complementary deoxyribonucleic molecules to which labeled deoxyribonucleic acid and ribonucleic acid molecules bind via sequence-specific hybridization.
Generally, radiometric or optical analysis of the microarray produces a scanned image consisting of a two-dimensional matrix, or grid, of pixels, each pixel having one or more intensity values corresponding to one or more signals. Scanned images are commonly produced electronically by optical or radiometric scanners and the resulting two-dimensional matrix of pixels is stored in computer memory or on a non-volatile storage device. Alternatively, analog methods of analysis, such as photography, can be used to produce continuous images of a microarray that can be then digitized by a scanning device and stored in computer memory or in a computer storage device.
Microarrays are often prepared on 1-inch by 3-inch glass substrates, not coincidentally having dimensions of common glass microscope slides. Commercial microarrays are often prepared on smaller substrates that are embedded in plastic housings. FIG. 1 shows a common, currently available commercial microarray packaged within a plastic housing. The microarray substrate 101 is embedded within the large, rather bulky plastic housing 102 to form an upper transparent cover over an aperture 103 within the plastic housing 102. The features that together compose the microarray are arranged on the inner, or downward surface of the substrate 101, and are thus exposed to a chamber within the plastic housing 102 comprising the microarray substrate 101 and the sides of the aperture 104-107. A transparent bottom cover may be embedded in the lower surface of the plastic housing to seal the chamber in order to create a small reaction vessel into which sample solutions may be introduced for hybridization with molecular species bound to the substrate of the microarray. Thus, the plastic housing serves to package the microarray and protect the microarray from contamination and mechanical damage during handling and storage and may also serve as a reaction chamber in which sample solutions are introduced for hybridization with features of the microarray. The plastic housing may further serve as a support for the microarray during optical or radiometric scanning of the microarray following exposure of the microarray to sample solutions. Scanning may, in certain cases, be carried out through the substrate of the microarray without a need to remove the microarray from the plastic housing.
Although currently commonly used and widely commercially available, the plastic microarray packaging shown in FIG. 1 has a number of disadvantages. First, it is necessary to seal the substrate of the microarray within the plastic housing to prevent exchange of liquids and vapors between the external environment and the reaction chamber formed by the substrate of the microarray, the plastic housing, and a bottom cover. Microarray substrates are commonly made from glass. Thus, a tight seal between the glass microarray substrate and the plastic housing is required. Unfortunately, many sealants used to seal glass to plastic may contain unreactive monomer or produce reactive surfaces that interfere chemically within the hybridization processes that need to be carried out within the reaction vessel. A second disadvantage is that glass and plastic exhibit different thermal expansion behaviors, creating high stress that may lead to glass-to-plastic bond failures during exposure of the plastic microarray packaging and embedded microarray to thermal fluctuations. A third disadvantage of the plastic packaging shown in FIG. 1 is that the plastic packaging is generally insufficiently mechanically stable to allow for reliable automated positioning of the microarray within a scanning device. As a result, scanning devices need an auto-focusing feature or other additional electromechanical systems for positioning the microarray within the scanning device. A fourth disadvantage of the plastic packaging shown in FIG. 1 is that, when the embedded microarray is scanned without removing the microarray from the plastic packaging, the thickness of the microarray substrate or of the lower transparent cover, depending from which side of the package the microarray is scanned, must have a relatively precise and uniform thickness so that the microarray substrate or bottom cover is not a source of uncontrolled error during the scanning process. Manufacturing either the microarray substrate or bottom cover to the required precision and uniformity adds to the cost of the microarray/plastic housing module. In general, fully automated manufacture of the plastic housing and embedded microarray is both complex and difficult. A final disadvantage of the plastic packaging for the microarray shown in FIG. 1 is that the microarray/plastic housing module is primarily designed for individual handling, and lacks features that would facilitate automated positioning, hybridization, and scanning of the microarray/plastic housing modules.
In order to address the above described deficiencies of the commonly used plastic microarray housing shown in FIG. 1, microarray strips have been developed. A microarray strip is a linear sequence of regularly-spaced, tightly sealed reaction chambers that each contains a precisely positioned and oriented microarray. The microarray strip further includes tractor feed perforations or other regularly spaced mechanical or optical features that allow the microarray strip, and the microarray contained within the microarray strip, to be mechanically translated and precisely positioned within various automated electromechanical systems. A microarray strip may also serve as a sequence of economical and reliable storage chambers and as packaging for storing, handling, and transporting microarrays contained within the microarray strip. The microarray strip may be rolled onto drums for compact and reliable storage of microarrays.
FIG. 2 shows a microarray strip. The microarray strip 200 comprises a pocket strip 202 and cover strip 204. The microarray strip 200 in FIG. 2 is shown during manufacture as the cover strip 204 is being laid down along the top surface of the pocket strip 202 to create sealed reaction chambers 206-207. A microarray 208 has been inserted into a pocket 210 of the pocket strip 202 which will be next covered by the cover strip 204 during the manufacturing process. An additional empty pocket 212, into which a next microarray will be placed, is located to the left of pocket 210 containing microarray 208. Membrane septa 214-220 are affixed to the cover strip 204 over corner regions of the sealed reaction chambers 206 and 207 to provide resealable ports through which solutions can be introduced into, and extracted from, the sealed reaction chambers. The septa are positioned above two elongated wells 222 and 224 formed by gaps between edges of an embedded microarray 208 and the sides of a pocket 226 and 228. Note that each microarray is positioned to rest on two ledges 230 (second ledge obscured in FIG. 2) to leave a gap between the microarray and the bottom 232 of the pocket in which the microarray is placed. The two linear wells 222 and 224 and the gap between the bottom active surface of the microarray and the bottom of the pocket 232 form a single continuous volume within the pocket. The ledges 230 may be designed so that the top surface of the microarray is flush with the upper surface of the pocket strip 234 or, alternatively, may be designed so that the upper surface of the microarray is recessed within each pocket to leave a gap between the upper surface of the microarray and the cover strip 204 following heat sealing of the cover strip 204 to the pocket strip 202. Generally, the active surface of the embedded microarrays, to which features are bonded, is positioned downward, and is opposite from the side of the microarray adjacent to the cover strip in the sealed reaction chambers. Both edges of the pocket strip contain a linear, regularly-spaced sequence of tractor feed perforations such as tractor perforation 236. These perforations can be enmeshed with gear-like feed rollers of various different mechanical systems to allow for automated translation of the microarray strip in a direction parallel to the length of the microarray strip and can also provide for precise mechanical positioning of the embedded microarrays within a scanning device.
Many types of microarray strips can be designed and manufactured, and many different types of materials may be employed. For example, the pocket strip and cover strip may be made from acrylonytrile-butodiene-styrene (xe2x80x9cABSxe2x80x9d) plastic and can be continuously manufactured via a vacuform process. The ABS pocket strip and cover strip can be readily heat sealed to provide a reasonably liquid-and-vapor-impermeable barrier. Alternatively, the cover strip may be sealed to the pocket strip via an adhesive sealant or may be designed to allow for mechanical sealing by application of mechanical pressure. Alternatively, both the pocket strip and cover strip may be manufactured from a plastic/metal foil laminate or other materials that provide a more robust barrier to exchange of liquid and vapor between the sealed reaction chambers and the outside environment. The septa can be affixed either to the upper surface or to the lower surface of the cover strip, or can be embedded within the cover strip, and can be manufactured from many different types of materials. One type of septa are three-ply laminates comprising an interior elastomer layer sandwiched between two polyester layers.
Although many of the deficiencies identified above for the commonly available plastic microarray housing shown in FIG. 1 are resolved by the newer microarray strip technology shown in FIG. 2, problems can arise in microarray strips due to small gaps between the bottom active surfaces of the microarrays and the bottoms of the pockets that contain them. Because solution in this gap is relatively immobilized by surface tension effects, mixing and circulating solutions within the pockets to thoroughly expose the active surfaces of microarrays to the solutions can be a difficult task. One technique is to introduce air bubbles into the gaps, and move, rotate, or shake the microarray strips to cause the bubbles to move within the gaps. When a bubble moves within a gap, solution is displaced, and mixing occurs. However, bubble movement within the solution is often accompanied by laminar flow within the solution, which, lacking vortices and other solution-mixing phenomena, does not lead to efficient mixing and circulation. More problematic is that the solution conformation of biopolymers can be disrupted at air/solution interfaces, so that the presence of a moving bubble can lead to denaturation of both solvated and bound molecules. This technique is also difficult to apply in a controlled manner, due to difficulties in guaranteeing well-distributed patterns of bubble movement within the gaps. For these reasons, designers, manufacturers, and users of microarray strips have recognized a need for a method and system for efficient microarray strip solution circulating and mixing.
One embodiment of the present invention is a microarray strip pocket with roll pump features that together compose a roll pump within the microarray strip pocket. A roll pump circulates and mixes solution contained in the gap between the active surface of a microarray positioned within the microarray strip pocket and the bottom, inner surface of the microarray strip pocket. The roll pump features include shallow and deep vertical wells that contain equal levels of solution when the microarray strip is level. The shallow and deep vertical wells, the gap between the active surface of a microarray and the bottom, inner surface of the microarray strip pocket, and a gap between the surface of an inclined feature connecting the vertical wells and a cover strip form a continuous volume, or space, within the reaction chamber formed when the cover strip is bonded to the pocket strip. As the microarray strip is rotated about an axis perpendicular to the edges of the microarray strip and in a plane parallel to the broad surfaces of the microarray, solution moves from the deep vertical wells into the gap between the active surface of a microarray and the bottom, inner surface of the microarray strip, and, as a result, solution is displaced from the gap to the shallow vertical wells. The displaced solution flows from the shallow vertical wells along the inner surface of the cover strip and back to the deep vertical wells as rotation about the axis continues. With each complete rotation, a volume of solution determined, in part, by the height of the solution level in the deep vertical wells passes through the gap between the active surface of a microarray and the bottom, inner surface of the microarray strip. By continuously rotating the microarray strip, solution is circulated through the gap and mixed within the gap.